When the method of culturing mammalian cells became standard laboratory practice in the 1950's, cell biologists started off by culturing the cells on the surface of Petri dishes, where the cells would proliferate in two dimensions. Cells growing on a planar surface could be easily observed at a single, fixed focus of the light microscope. Nutrients in the form of tissue culture media could be easily replenished, with direct contact between the cells and media, and no mass transfer limitations. The cells could be conveniently replated or subcultured to other wells or dishes by trypsinizing the cells from the original surface, while leaving the latter intact. Indeed, the polished practice of 2-dimensional (2-D) culture has contributed to important discoveries in biology and medicine.
In terms of representing the true environment of the cells in vivo, however, it is becoming apparent that a 2-D cell culture is an over-simplification, if not a misrepresentation. With the exception of epithelial cells, most mammalian cells exist in vivo embedded in a 3-D substance or extracellular matrix (ECM). For example, the distribution of cytoskeletal elements in 3-D is vastly different from that in 2-D. Fibroblasts cultured in 2-D show a high concentration of stress fibers on the surface of the cell in contact with the plate, whereas in 3-D, these stress fibers are absent. In view of the fact that the biochemical signals that direct cell fate work in conjunction with biomechanical signals, it can be inferred that the more ‘natural’ response of a cell towards a biochemical stimuli (e.g. cytokine/drug) would be observed in 3 dimensions. It has been shown that cell adhesion, migration and phenotype in 3-D culture is quite different from 2-D cell culture. Cukierman et al. have also elucidated a mode of matrix adhesion unique to cells in 3-D culture (E. Cukierman, R. Pankov, D. R. Stevens, K. M. Yamada, Taking cell-matrix adhesions to the third dimension, Science, 294 (2001), 1708-1712). In this context, there is a need for systems that are specifically designed for 3-D cell and tissue culture.
The existing 3-D cell culture systems are mainly based on photo-crosslinkable acrylate-based polymers, self-assembled peptides, and matrix-metalloproteinase (MMP) degradable systems. New systems continued to be developed, reflecting the keen interest in this field. However, none of these systems possess the property of reversible gelation.
Existing technology for forming gels for 3-D cell culture relies on the use of chemical crosslinkers, photo-initiators, ultraviolet light or a change in ionic strength for the crosslinking of gels. Gelation based on chemical reaction must be performed with care, since the reaction could be detrimental to the cells and proteins within the gel. Most of the commonly used protein-based gels (e.g. Matrigel and collagen Type I) rely on a change in temperature to induce gelation. This entails the inconvenience of storing the gel precursor at a low temperature, as well as inconsistencies in the setting time of the gel (depending on the cell culture vessel and volume subjected to the temperature change).
There is therefore a need for a gel which is reversibly formable by a convenient and robust means.